Project Summary Proper control of gene expression is essential for life. While substantial advances have been made in the discovery of DNA sequences and transcription factors that act combinatorially to regulate transcription, much less is known about later steps in the gene expression program. Nevertheless, it is now clear that substantial regulation of gene expression occurs post-transcriptionally, in pre-mRNA splicing, RNA localization, translation, and RNA decay, and in the coordination of RNAs by RNA binding proteins (RBPs), microRNAs, and RNA chemical modifications. While many sequence motifs are known to mediate post-transcriptional regulation, RNA secondary structures that govern access to these motifs are much less well understood. The long term goal of this project is to enable structural characterization of RNAs on a genome-wide scale. First, we will apply a recently developed method to map secondary structures of most human RNAs in living cells. Second, we will develop methods to follow the fate of RNA secondary structures through the RNA life cycle. Third, we will develop high throughput methods to perturb and test functions of RNA structures. This integrated pipeline of new technologies will enable investigators to identify and decode regulatory RNA elements in the genome with unprecedented speed and accuracy.